Recent advances in the development of carbohydrate based therapeutics (Koeller and Wong, Nat. Biotechnol., 18 (2000) 835-841), and the limitations of present chemical synthetic methods for producing oligosaccharides, has led to more novel approaches to the synthesis of carbohydrates and their conjugates (Davis, J. Chem. Soc. Perkin Trans., 1 (2000) 2137). One approach to this problem is to carry out such syntheses using carbohydrate processing enzymes such as glycosyltransferases or glycosidases, as a valuable source of catalytic activity for the manipulation of unprotected carbohydrates (Crout and Vic, Curr. Opin. Chem. Biol., 2 (1998) 98-11); Wymer and Toone, Curr. Opin. Chem. Biol., 4 (2000) 110-119; Watt et al., Curr. Opin. Chem. Biol., 7 (1997) 652-660; Kren and Thiem, Chem. Soc. Rev., 26 (1997) 463-473; and Palcic, Curr. Opin. Biotechnol., 10 (1999) 616-624). Glycosidases are simple, robust, soluble enzymes, and in general have been preferred for such glycosynthesis (Scigelova et al., J. Mol. Catal. B Enzym., 6 (1999) 483-494 and Van Rantwijk et al., J. Mol. Catal. B Enzym., 6 (1999) 511-532). Although catalysis of the hydrolysis of glycoside bonds is normally observed, glycosidases may be successfully used to synthesise glycosides through reverse hydrolysis (thermodynamic control) or transglycosylation (kinetic control with activated donors) strategies.
Thus far, improvements in glycosidase synthetic utility have largely focused upon developing new strategies for increasing low product yields (Mackenzie et al., J. Am. Chem. Soc., 120 (1998) 5583-5584), improving regioselectivity of transfer (Prade et al., Carbohydr. Res., 305 (1998) 371-381) or characterising available glycosidases for novel activities (Scigelova et al., supra). For example, a major advance in improving yields has been the development of the glycosynthase by Withers and co-workers (Mackenzie et al., supra; Mayer et al., FEBS Lett., 466 (2000) 40-44, Malet and Planas, FEBS Lett., 440 (1998) 208-212; Moracci et al., Biochemistry 37 (1998) 17262-17270, Trincone and Perugino, Bioorg. Med. Chem. Lett., 10 (2000) 365-368; Fort et al., J. Am. Chem. Soc., 122 (2000) 5429-5437; and Nashiru et al., Chem. Int. Ed., 40 (2001) 417-420). These nucleophile-less glycosidase mutants are capable of glycosyl transfer in yields of up to 90% using glycosyl fluoride donors, but do not hydrolyse glycoside products and they illustrate well the benefits of glycosidase engineering for creating more synthetically useful catalysts.
An area of glycosidase engineering which has thus far been largely neglected is the engineering of new substrate specificities (Zhang et al., Proc. Natl. Acad. Sci. USA., 94 (1997) 4504-4509; Andrews et al., J. Biol. Chem., 275 (2000) 23027-23033; Kaper et al., Biochemistry 39 (2000) 4963-4970; and Rye and Withers, Curr. Opin. Chem. Biol., 4 (2000) 573-580). Since the nature of the parent carbohydrate to be coupled to a given acceptor may be determined in synthesis simply through appropriate choice of donor, it is largely the stereoselectivity of a given glycosidase that we wish to exploit. An area of growing interest is that of combinatorial biocatalysis: the use of enzyme catalysts in parallel reactions to provide arrays of related molecules (Michels et al., Trends Biotechnol., 16 (1998) 210-215; and Krstenansky and Khmelnitsky, Bioorg. Med. Chem., 7 (1999) 2157-2162). In particular, the importance of gaining access to diverse arrays of glycoconjugates has recently been highlighted (Barton et al., Nat. Struct. Biol., 8 (2001) 545-551). However, although combinatorial chemistry has revolutionized the approach to traditional chemical synthesis, the development of combinatorial biocatalysis has been hampered by the often stringent substrate specificities of synthetically useful enzymes.